Modified bacteria and uses thereof

Abstract

Provided are modified Gram-negative bacteria having an increased periplasmic volume. Also provided are methods of expressing exogenous genes in the bacteria and targeting protein production to the periplasmic space.

1 . A recombinant, Gram-negative, bacterial cell having a periplasm and a cytoplasm, wherein the ratio of the periplasmic volume to the cytoplasmic volume is between about 0.5:1 and about 10:1, wherein the bacterial cell comprises an exogenous gene encoding a protein of interest. 
     
     
         2 . The bacterial cell of  claim 1 , wherein the bacterium is selected from: gammaproteobacteria and alphaproteobacteria. 
     
     
         3 . The bacterial cell of  claim 2 , wherein the bacterium is selected from:
 Escherichia coli, Vibrio natriegens, Pseudomonas fluorescens, Caulobacter crescentus, Agrobacterium tumefaciens, and Brevundimonas diminuta.   
     
     
         4 . The bacterial cell of  claim 3 , wherein the bacterium is Escherichia coli. 
     
     
         5 . The bacterial cell of  claim 4 , wherein the bacterium is Escherichia coli BL21(DE3). 
     
     
         6 . The bacterial cell of  claim 1 , wherein the ratio of the periplasmic volume to the cytoplasmic volume is between about 0.5:1 and about 5:1. 
     
     
         7 . The bacterial cell of  claim 1 , wherein the ratio of the periplasmic volume to the cytoplasmic volume is between about 0.5:1 and about 1:1. 
     
     
         8 . The bacterial cell of  claim 1 , wherein the ratio of the periplasmic volume to the cytoplasmic volume is between about 1:1 and about 10:1. 
     
     
         9 . The bacterial cell of  claim 1 , wherein the ratio of the periplasmic volume to the cytoplasmic volume is between about 1:1 and about 5:1. 
     
     
         10 . The bacterial cell of  claim 1 , wherein the ratio of the periplasmic volume to the cytoplasmic volume is between about 5:1 and about 10:1. 
     
     
         11 . The bacterial cell of  claim 1 , wherein the bacterial cell is a coccus having a longest dimension of about 2 µm to about 16 µm. 
     
     
         12 . The bacterial cell of  claim 11 , wherein the longest dimension is about 4 µm to about 16 µm. 
     
     
         13 . The bacterial cell of  claim 11 , wherein the longest dimension is about 8 µm to about 16 µm. 
     
     
         14 . The bacterial cell of  claim 11 , wherein the longest dimension is about 2 µm to about 8 µm. 
     
     
         15 . The bacterial cell of  claim 11 , wherein the longest dimension is about 4 µm to about 8 µm. 
     
     
         16 . The bacterial cell of  claim 11 , wherein the longest dimension is about 2 µm to about 4 µm. 
     
     
         17 . The bacterial cell of  claim 1 , wherein the bacterial cell comprises an expression vector comprising the exogenous gene. 
     
     
         18 . The bacterial cell of  claim 17 , wherein the expression vector is free of a marker encoding for resistance to an inhibitor of bacterial cell peptidoglycan biogenesis. 
     
     
         19 - 33 . (canceled) 
     
     
         34 . A cell culture comprising the bacterial cell of  claim 1  and a medium comprising a magnesium salt, wherein the concentration of magnesium ions in the medium is at least about 4 mM,. 
     
     
         35 - 98 . (canceled) 
     
     
         99 . A fermentation vessel containing a cell culture of  claim 34  any, wherein the cell culture comprises a volume of medium of about, or of at least about, 1 L; 10 L; 100 L; 250 L; 500 L; or 1,000 L.